This study was approved by the Institutional Review Committee on the Ethics of Animal
Experiments of Iwate Medical University. All experiments were conducted in accordance
with the Institutional Animal Care and Use Committee guidelines (Ethical number is
26-010).
Experimental materials
The pigs are slaughtered as part of routine procedure in a slaughter house. We obtained
the lung of the pig from this slaughter house.
The
third of pulmonary arteries, which were 2 to 3 mm in diameter, were excised from
the lung of a slaughtered pig (6 months of age) and cut into rings 2 to 3 mm in length.
The endothelium, which was rubbed gently against the thin arm of stainless steel tweezers,
was then denuded and the rings inverted to prepare specimens of pulmonary artery vascular
smooth muscle, with the inner surface presented outward. It was confirmed that 3µM
acetylcholine-induced relaxation of the artery rings disappeared after this procedure. N
means number of rings.
Experimental methods
Method for isometric contraction tension
Specimens were placed in the incubator (1.0 mL) and a resting tension of 4 mN was
applied. After perfusion with the Hank’s balanced salt solution (HBSS) for 30 min,
various stimulants were administered; the resulting contraction tension was simultaneously
measured. To measure contraction tension, one end of the specimen in the incubator
was fixed to a manipulator (M-152; NARISHIGE, Tokyo, Japan ) and the other end was
fixed via a tungsten wire to a tension transducer (UL-2GR; Minebea, Tokyo, Japan).
Data were recorded on PowerLab® (ADInstruments, Bella Vista, Australia) via a pressure amplification unit (N4438;
NEC San-ei, Tokyo, Japan). The composition of HBSS was as follows: 0.34 mM Na2HPO4·7H2O, 0.44 mM KH2PO4, 0.8 mM MgSO4, 1.26 mM CaCl2, 4.2 mM NaHCO3, 5.55 mM glucose, 5.36 mM KCl, and 137 mM NaCl at pH 7.37.
Effects of various α receptors on the vascular smooth muscle of pulmonary arteries
Direct effects of various concentrations of Dex and imidazoline on vascular smooth
muscle
After an approximate 2-min perfusion with 60 mM KCl solution, contraction tension
was measured, recorded, and used as control values. Next, HBSS containing 10−10, 10−9, 10−8, 10−7, 10−6, 5 × 10−6, and 10−5 M Dex (Wako; molecular weight, 266.55, Tokyo, Japan) or imidazoline (Wako; molecular
weight, 236.74, Tokyo, Japan) were administered for 2 min each, in that order. Changes
in the resulting isometric contraction tension at each concentration were measured
and recorded (Fig. 1).
Effects of various concentrations of Dex and imidazoline on high KCl-induced contraction
tension
After an approximate 2-min perfusion with 60 mM KCl solution, contraction tension
was measured, recorded, and used as control value. Next, 60 mM KCl containing 10−10, 10−9, 10−8, 10−7, 10−6, 5 × 10−6, and 10−5 M Dex or imidazoline were administered for 2 min each, in that order. Change in isometric
contraction tension at each concentration was measured and recorded and dose dependence
curve was constructed (Fig. 2).
Effects of yohimbine, rauwolscine, and prazosin on high KCl-induced contraction tension
After an approximate 2-min perfusion with 60 mM KCl solution, contraction tension
was measured, recorded, and used as control value. Next, 60 mM KCl solution containing
5 × 10−6 M Dex was administered, and changes in contraction tension was measured and recorded.
Then 60 mM KCl solutions containing 5 × 10−6 M yohimbine, rauwolscine, or prazosin were administered for 2 min each, and change
in contraction tension was measured and recorded (Fig. 3).
Effects of various concentrations of Dex, imidazoline, yohimbine, and rauwolscine
on adrenaline-induced contraction tension
After an approximate 2-min perfusion with HBSS containing 5 × 10−6 M adrenaline (Wako; molecular weight, 333.6, Tokyo, Japan), contraction tension was
measured, recorded, and used as control value. Next, 5 × 10−6 M adrenaline solutions containing 10−10, 10−9, 10−8, 10−7, 10−6, 5 × 10−6, and 10−5 M Dex
, imidazoline, yohimbine, and rauwolscine were administered for 2 min each, in that
order. Change in contraction tension at each concentration was measured and recorded
(Fig. 4).
Effects of Dex on adrenaline-induced contraction tension
with [Ca2+]i reservoir depletion
After an approximate 2-min perfusion with 60 mM KCl solution, contraction tension
was measured, recorded, and used as control value. Next, under perfusion with a Ca2+-free HBSS containing 1 × 10−3 M EDTA, the following drugs were administered: 2.5 × 10−2 M caffeine (Wako: molecular weight, 212.21, Tokyo, Japan) was administered for 2
min, 3 × 10−5 M ryanodine (Wako: molecular weight, 493.55, Tokyo, Japan) was administered for 5
min, and then 2.5 × 10−2 M caffeine was administered twice for 2 min each. Subsequently,
a Ca2+-containing HBSS containing 5 × 10−6 M adrenaline or Ca2+-containing HBSS containing 5 × 10−6 M adrenaline supplemented with 5 × 10−6 M Dex was administered for approximately 30 min
. Change in contraction tension was measured and recorded (Fig. 5).
Effects of Dex on adrenaline-, caffeine- and histamine-induced contraction tension
in the absence of extracellular Ca2+
After an approximate 2-min perfusion with 60 mM KCl solution, contraction tension
was measured, recorded, and used as control value. Next, after 15 min of perfusion
with a Ca2+-free HBSS containing 1 × 10−3 M EDTA, 5 × 10−6 M adrenaline and histamine, and 25 mM caffeine were administered for 2 min, and contraction
tension was measured and recorded. Next, reference values were measured in the same
manner as control values, 5 × 10−6 M adrenaline and histamine, and 25 mM caffeine supplemented with 5 × 10−6 M Dex were administered for 2 min after 30 min of perfusion with a Ca2+-free HBSS containing 1 × 10−3 M EDTA, and then contraction tension was measured and recorded
(Fig. 6).
Statistical Analysis
Values are presented as mean ± SEM. Statistical analysis was performed with SPSS,
version 11.0 (SPSS, Chicago, IL, USA). Differences between the means of two groups
were evaluated with Student’s
t test. Differences among multiple groups, whose homogeneity of variance was assessed
with Levene’s test, were evaluated with repeat measure analysis of variance (ANOVA)
followed by Scheffe’s multiple comparison procedure. Differences were considered significant
at p-values of <0.05.